A spectrum of human neurological disorders including epilepsies, Lissencephaly or pediatric cancer is due in part to defective neuronal motility or germinal zone (GZ) exit and the resultant errors in neuronal circuit formation. To design strategies to prevent or treat such disorders, the field has sought to clarify the molecular mechanisms regulating neuronal motility and migration initiation. Despite advances implicating various genes essential for neuronal migration, a key gap in our knowledge is to discover how disparate cytoskeletal or signaling molecules cooperatively execute complex neuronal motility programs, such as GZ exit or nucleokinesis. My laboratory has tackled this challenge by dissecting neuronal polarity pathways impacting neuronal differentiation, nucleokinesis and adhesion control during GZ exit using the evolutionarily conserved Partitioning Defective, or Pard, polarity signaling complex as a molecular entry point. We gained insights into the regulatory logic controlling polarity during cerebellar granule neuron (CGN) development by discovering that the Seven in Absentia 2 (Siah2) E3 ubiquitin ligase is a Pard, complex antagonist. Siah2 is heavily expressed in CGN progenitors (GNPs); but not postmitotic CGNs, where Siah2-targeting of Pard3 for degradation constitutes an active pathway for progenitor polarity inhibition. We are uniquely positioned to discover new cellular mechanisms that control the onset of neuronal polarity since others overlooked inhibitory pathways in the past. In preliminary studies, we characterized new Siah2 targets relevant to GZ exit and radial migration: the Deleted in Colorectal Carcinoma (DCC) Netrin-1 (Ntn1) receptor and drebrin microtubule-actin crosslinking protein. Preliminary analysis shows: 1) Ntn1 stimulates CGN GZ exit and that Siah2 inhibits, while Pard3 promotes Ntn1-induced DCC receptor exocytosis, suggesting a hypothesis that Siah2-Pard3 antagonism regulates CGN sensitivity to Ntn1 GZ repulsion possibly through a link between DCC and junctional adhesion molecule-C (JAM-C), an adhesion receptor exocytosed in a Pard3-dependent manner. 2) Drebrin links actin- microtubule dynamics that are in turn regulated by Siah2 ubiquitination, suggesting a hypothesis that Siah2- drebrin antagonism governs the onset of nucleokinesis via microtubule-actin interactions. Remarkably, Siah2 expression is enhanced in Lissencephaly 1 (Lis1) deficient CGNs and Siah2 loss of function (LOF) or drebrin gain of function (LOF) rescues Lis1LOF migration phenotypes, suggesting that altering the balance of microtubule-actin interactions could have therapeutic value in classic neuronal migration disorders. We will build on our expertise examining polarity signaling in neuronal migration to combine in vivo genetics and ex vivo mechanistic studies with the power of transformative imaging technologies like Lattice Light Sheet Microscopy (LLSM) live-cell imaging to explore the following aims: Aim1: Define how Siah2 and Pard3 regulate DCC-dependent Ntn1 GZ repulsion. Challenge: Our current understanding of how guidance cues are interpreted in conjunction with cell adhesion and neuronal polarity pathways is limited. The findings that Siah2-Pard3 antagonism regulates DCC trafficking through a link to JAM-C adhesion sites presents an unique opportunity to test the premise that an adhesion and guidance receptor coincidence detection circuit control migration initiation. Approach: We will ablate DCC and Ntn1 in GNPs to confirm GZ repulsion in vivo. We will use epistasis in ex vivo slices or Ntn1 gradients in combination with mechanistic live-cell imaging, including LLSM, to assess how Ntn1 sensitivity couples to migration path selection via Siah2 antagonism of Pard3, DCC and JAM-C. Impact: Aim 1 will provide a new conceptual model for how polarity inhibition regulates GNP GZ occupancy and how relief of inhibition in postmitotic CGN stimulates GZ exit through adhesion and guidance receptors. Aim 2: Determine how Siah2 regulates microtubule-actin interactions during neuronal differentiation and Lis1-deficient CGNs. Challenge: Our current understanding of how cytoskeletal systems cooperate to execute radial migration or how these interactions fail in migration disorders is poor. Our findings that Siah2 regulates drebrin-dependent microtubule-actin interactions and Siah2LOF rescues Lis1LOF migration defects presents an opportunity to test two premises: 1) enhanced cytoskeletal interactions during CGN differentiation regulate the onset of classic nucleokinesis and 2) enhanced microtubule-actin restore migration in Lis1-deficient CGNs. Approach: We will use in vivo genetics, an ex vivo epistatis screen and live-cell imaging assays to assess how Siah2 mechanistically controls microtubule-actin interactions in normal or pathologic forms of CGN migration. Impact: Aim 2 could provide a new conceptual model of how the cytoskeletal interactions that drive neuronal motility are elaborated when neuronal progenitors transition to postmitotic neurons and may open the potential to exploit these mechanisms to further understand neuronal migration disorders. The proposed studies will also provide key new insights into addition questions: 1) what are the cell biological pathways that work in parallel to classic cell polarity signaling pathway during neural development and 2) how are rapid cell biological responses during brain controlled by post-translational processes like ubiquitination.